Neural stem cells (NSCs) in the adult brain transit from the quiescent state to proliferation to produce new neurons. The mechanisms regulating this transition in freely behaving animals are, however, poorly understood. We customized in vivo imaging protocols to follow NSCs for several days up to months, observing their activation kinetics in freely behaving mice. Strikingly, NSC division is more frequent during daylight and is inhibited by darkness-induced melatonin signaling. The inhibition of melatonin receptors affected intracellular Ca2+ dynamics and promoted NSC activation. We further discovered a Ca2+ signature of quiescent versus activated NSCs and showed that several microenvironmental signals converge on intracellular Ca2+ pathways to regulate NSC quiescence and activation. In vivo NSC-specific optogenetic modulation of Ca2+ fluxes to mimic quiescent-state-like Ca2+ dynamics in freely behaving mice blocked NSC activation and maintained their quiescence, pointing to the regulatory mechanisms mediating NSC activation in freely behaving animals.The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a GGGGCC repeat expansion in the C9orf72 gene. We developed a platform to interrogate the chromatin accessibility landscape and transcriptional program within neurons during degeneration. We provide evidence that neurons expressing the dipeptide repeat protein poly(proline-arginine), translated from the C9orf72 repeat expansion, activate a highly specific transcriptional program, exemplified by a single transcription factor, p53. Ablating p53 in mice completely rescued neurons from degeneration and markedly increased survival in a C9orf72 mouse model. p53 reduction also rescued axonal degeneration caused by poly(glycine-arginine), increased survival of C9orf72 ALS/FTD-patient-induced pluripotent stem cell (iPSC)-derived motor neurons, and mitigated neurodegeneration in a C9orf72 fly model. We show that p53 activates a downstream transcriptional program, including Puma, which drives neurodegeneration. These data demonstrate a neurodegenerative mechanism dynamically regulated through transcription-factor-binding events and provide a framework to apply chromatin accessibility and transcription program profiles to neurodegeneration.Epstein-Barr virus (EBV) is an oncogenic human herpesvirus that persists as a multicopy episome in proliferating host cells. Episome maintenance is strictly dependent on EBNA1, a sequence-specific DNA-binding protein with no known enzymatic activities. Here, we show that EBNA1 forms a cell cycle-dependent DNA crosslink with the EBV origin of plasmid replication oriP. EBNA1 tyrosine 518 (Y518) is essential for crosslinking to oriP and functionally required for episome maintenance and generation of EBV-transformed lymphoblastoid cell lines (LCLs). Mechanistically, Y518 is required for replication fork termination at oriP in vivo and for formation of SDS-resistant complexes in vitro. EBNA1-DNA crosslinking corresponds to single-strand endonuclease activity specific to DNA structures enriched at replication-termination sites, such as 4-way junctions. These findings reveal that EBNA1 forms tyrosine-dependent DNA-protein crosslinks and single-strand cleavage at oriP required for replication termination and viral episome maintenance.This year marks the 25th year of publication for Nursing for Women's Health.Cranial neural crest (CNC) cells give rise to bone, cartilage, tendons, and ligaments of the vertebrate craniofacial musculoskeletal complex, as well as regulate mesoderm-derived craniofacial muscle development through cell-cell interactions. Using the mouse soft palate as a model, we performed an unbiased single-cell RNA-seq analysis to investigate the heterogeneity and lineage commitment of CNC derivatives during craniofacial muscle development. We show that Runx2, a known osteogenic regulator, is expressed in the CNC-derived perimysial and progenitor populations. Loss of Runx2 in CNC-derivatives results in reduced expression of perimysial markers (Aldh1a2 and Hic1) as well as soft palate muscle defects in Osr2-Cre;Runx2fl/fl mice. We further reveal that Runx2 maintains perimysial marker expression through suppressing Twist1, and that myogenesis is restored in Osr2-Cre;Runx2fl/fl;Twist1fl/+ mice. Collectively, our findings highlight the roles of Runx2, Twist1, and their interaction in regulating the fate of CNC-derived cells as they guide craniofacial muscle development through cell-cell interactions.Enzymes represent a significant proportion of the druggable genome and constitute a rich source of drug targets. https://www.selleckchem.com/products/lanifibranor-iva-337.html Delivery of a successful program for developing a modulator of enzyme activity requires an understanding of the enzyme's mechanism and the mode of interaction of compounds. This allows an understanding of how physiological conditions in disease-relevant cells will affect inhibitor potency. As a result, there is increasing interest in evaluating hit compounds from high-throughput screens to determine their mode of interaction with the target. This work revisits the common inhibition modalities and illustrates the impact of substrate concentration relative to Km upon the pattern of changes in IC50 that are expected for increasing substrate concentration. It proposes a new, high-throughput approach for assessing mode of inhibition, incorporating analyses based on a minimal descriptive model, to deliver a workflow that allows rapid and earlier compound classification immediately after high-throughput screening.For nearly two decades mass spectrometry has been used as a label-free, direct-detection method for both functional and affinity-based screening of a wide range of therapeutically relevant target classes. Here, we present an overview of several established and emerging mass spectrometry platforms and summarize the unique strengths and performance characteristics of each as they apply to high-throughput screening. Multiple examples from the recent literature are highlighted in order to illustrate the power of each individual technique, with special emphasis given to cases where the use of mass spectrometry was found to be differentiating when compared with other detection formats. Indeed, as many of these examples will demonstrate, the inherent strengths of mass spectrometry-sensitivity, specificity, wide dynamic range, and amenability to complex matrices-can be leveraged to enhance the discriminating power and physiological relevance of assays included in screening cascades. It is our hope that this review will serve as a useful guide to readers of all backgrounds and experience levels on the applicability and benefits of mass spectrometry in the search for hits, leads, and, ultimately, drugs.